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High Throughput Proteomic Applications Using Anti‐DYKDDDDK Magnetic Agarose
Author(s) -
Benton Betsy,
Geddes Joanna,
Vattem Krishna,
Patel Bhavin,
Kaboord Barbara
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.802.1
Subject(s) - agarose , immunoprecipitation , microbiology and biotechnology , chemistry , proteomics , protein purification , epitope , hela , computational biology , in vitro , mass spectrometry , biology , biochemistry , chromatography , antibody , immunology , gene
Affinity purification, immunoprecipitation and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti‐DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against DYKDDDDK tag covalently attached to high binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen™ ExpiCHO Expression System and by a HeLa in vitro translation (cell‐free) system. Biological activity was demonstrated for ExpiCHO‐expressed DYKDDDDK‐Tumor necrosis factor alpha (TNFa) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB). In addition, successful co‐immunoprecipitation of DYKDDDDK‐tagged BAD protein with 14‐3‐3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific KingFisher Flex magnetic particle processor and mass spectrometry. In summary, anti‐DYKK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .